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Rho Inhibitor I

Rho Inhibitor I

*Amount

1x20 ug (Cat. # CT04-A) +$185.00
5x20 ug (Cat. # CT04-B) +$525.00
20x20 ug (Cat. # CT04-C) +$1,450.00

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Product Uses Include

Inhibit Rho activity within 4h (compared to 24h with siRNA)
Stop Rho pathway signalling
Determine "activator" specificity for Rho pathway
Positive control for Rho Inhibition in cell morphology studies
Positive control for Rho inhibition in cell extract biochemical studies
Inhibit stress fiber formation
Inhibit RhoA, RhoB and RhoC in living cells
Inhibits Rho in all cell types currently tested

Material
This product consists of highly purified C3 Transferase (Cat. # CT03) covalently linked to a proprietary cell penetrating moiety via a disulfide bond. The cell penetrating moiety allows rapid and efficient transport through the plasma membrane. Once in the cytosol, the cell penetrating moiety is released, thereby allowing C3 Transferase to freely diffuse intracellularly and inactive RhoA, RhoB, and RhoC, but not related GTPases such as Cdc42 or Rac1.

The Exoenzyme C3 Transferase from Clostridium botulinum is commonly used to selectively inactivate the GTPases RhoA, RhoB, and RhoC, both in vivo and in vitro. C3 Transferase inhibits Rho proteins by ADP-ribosylation on asparagine 41 in the effector binding domain of the GTPase. A major limitation of C3 Transferase for use in in vivo applications is that this protein is only slightly cell permeable. Consequently, overnight incubations with C3 Transferase at concentrations as high as 100 µg/ml are often necessary to inactivate Rho proteins in cultured cells. Under these conditions, the long incubation period and large amount of C3 Transferase that are required can be disruptive to basic cellular functions and economically burdensome. Cytoskeleton, Inc. has overcome these problems by providing a cell permeable form or C3 Transferase that can efficiently inactivate cellular Rho proteins in as little as 2 h.

CT04 has been used to inactive Rho proteins to an efficiency of 75-95% in fibroblasts, neurons, epithelial, endothelial, and hematopoietic cells as well as other primary and immortalized cell lines (see Table 1 for recommended conditions of use for different cells types).

Table 1. Suggested Conditions for Rho inactivation by Cell Permeable C3 Transferase. The indicated cells were subjected to Rho inactivation assays with CT04. Note: These concentrations were determined in serum free medium. For cells grown in serum containing medium the recommended concentration is four times higher.
Cell Type	Cell Line	Moderate phenotype*	Robust phenotype**
Fibroblast	Swiss 3T3	2.0 µg/ml, 2 h^{(1, 2)}	2.0 µg/ml, 4-6 h^{(1, 2)}
Epithelial	HeLa	0.5 µg/ml, 2 h^{(1, 2)}	0.5 µg/ml, 4-6 h^{(1, 2)}
Epithelial	MDCK	1.0 µg/ml, 2 h^{(1, 2)}	1.0 µg/ml, 4-6 h^{(1, 2)}
Endothelial	HUVEC	1.0 µg/ml, 2 h^{(1, 2)}	1.0 µg/ml, 4-6 h^{(1, 2)}
Hematopoietic	Jurkat	1.0 µg/ml, 2 h⁽²⁾	1.0 µg/ml, 4 h⁽²⁾
Neuronal	Mouse cortex	1.0 µg/ml, 4 h⁽²⁾	1.0 µg/ml, 4-6 h^{(1, 2)}
* A moderate phenotype is characterized by a 10-40% decrease in Rho activity accompanied by stress fiber disruption and a well spread morphology (see Figures 1 and 2). ** A robust phenotype is characterized by a >50% decrease in Rho activity accompanied by a loss of stress fibers, cell body collapse, and protrusion of dendrite-like extensions (see Figures 1 and 2). ¹ Based on morphological assays examining cell shape and the architecture of the actin cytoskeleton. ² Based on biochemical data from Rho activity assays.

Purity
The Exoenzyme C3 Transferase from Clostridium botulinum has been produced in a bacterial expression system. The recombinant protein contains six histidine residues at its amino terminus (His tag), has a molecular weight of approximately 24 kDa, and is greater than 90% pure (see Cat. # CT03 for gel purity). To make the purified C3 Transferase protein cell permeable, a proprietary cell penetrating moiety has been linked via a reversible disulfide bond. Cell Permeable Rho Inhibitor is supplied as a white lyophilized powder.

Biological Activity
Cell Permeable Rho Inhibitor (Cat. # CT04) is useful for efficient inactivation of RhoA, RhoB, and RhoC in a variety of cultured cells. The reagent inhibits Rho proteins in fibroblasts, neurons, epithelial, endothelial, and hematopoietic cells as well as other primary and immortalized lines. Cells treated with Cell Permeable C3 Transferase can be subjected to any one of a number of assays that indicate a decrease in Rho activity, including focal adhesion or stress fiber (Cat. # BK005) disruption assays and Rho activity assays by G-LISA™ (Cat. # BK124) or pulldown (Cat. # BK036). See Figures 1 and 2 for examples of stress fiber disruption and Rho inactivation demonstrating CT04 biological activity.


	Figure 1. Cell permeable Rho inhibitor disrupts stress fibers and can be manipulated to induce either moderate or robust phenotypes. Swiss 3T3 fibroblasts plated on coverslips were untreated (A) or treated with 2.0 µg/ml of CT04 for 2 h (B) or 4 h (C) at 37°C. Cell were then fixed, stained with Rhodamine-labeled Phalloidin (Cat. # BK005), and visualized by flu orescence microscopy. Images were taken at a magnification of 20×. The untreated control cells in A were well spread and stress fibers were present. The cells treated for 2 h in B displayed a Moderate Phenotype, characterized by a loss of stress fibers, cells remaining well spread, and a 10-40% decrease in Rho activity (also see Figure 2). Treatment for 4 h (C) yielded a Robust Phenotype, characterized by a loss of stress fibers, decreased cell spreading, collapse of the cell body, protrusion of dentritic extensions, and a >50% decrease in Rho activity (also see Figure 2).


	Figure 2a. CT04 inhibition of Rho activity as measured with the RhoA G-LISA Activation Assay (Cat.# BK124). Serum starved Swiss 3T3 fibroblasts were untreated (no CT04) or trea ted with 0.20, 0.50 and 2.0 µg/ml of CT04 for 4h in serum free medium at 37°C, then activated with 100µg/ml calpeptin for 10min. Cells were then lysed and RhoA activity was measured by the RhoA G-LISA Activation Assay (Cat.# BK124). Note: At 2.0 µg/ml CT04 for 4h results in almost complete (90%) inhibition of RhoA activity.


	Figure 2b. Cell permeable Rho Inhibitor decreases RhoA activity. Swiss 3T3 fibroblasts were untreated (no CT04) or treated with 2.0 µg/ml of CT04 for 2 h (CT04, 2 h) or 4 h (CT04, 4 h) at 37°C. Cells were then lysed and RhoA activity was measured by pulldown assay using the Rho-binding domain of the Rho effector Rhotekin (RhoA Activation Assay Biochem Kit, Cat. # BK036). The pulldowns (active RhoA) and cell extracts (total RhoA) were analyzed by SDS-PAGE followed by Western blotting with a RhoA specific antibody. The level of RhoA activity in each sample is proportional to the amount of RhoA precipitated in the pulldowns (active RhoA, upper panel).

The concentration of Cell Permeable C3 Transferase required for efficient inactivation of Rho proteins can vary between cell types. In addition, the length of treatment can be manipulated to yield a moderate or robust phenotype (see Figures 1 and 2). For these reasons, the concentration of this reagent and the duration of treatment should be determined by the user. Typically the effective range is between 0.5 and 2.0 µg/ml with incubations times of 2-4 h for a moderate phenotype and 4-6 h for a robust phenotype. Recommended conditions for several cell types are detailed in Table 1.

Rho Inhibitor I

Rho Inhibitor I

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